Progression-free survival and overall survival after BRCA1/2-associated epithelial ovarian cancer: A matched cohort study

Introduction Germline BRCA1/2-associated epithelial ovarian cancer has been associated with better progression-free survival and overall survival than sporadic epithelial ovarian cancer, but conclusive data are lacking. Methods We matched 389 BRCA1-associated and 123 BRCA2-associated epithelial ovarian cancer patients 1:1 to sporadic epithelial ovarian cancer patients on year of birth, year of diagnosis, and FIGO stage (< = IIA/> = IIB). Germline DNA test was performed before or after epithelial ovarian cancer diagnosis. All patients received chemotherapy. We used Cox proportional hazards models to estimate the associations between mutation status (BRCA1 or BRCA2 versus sporadic) and progression-free survival and overall survival. To investigate whether DNA testing after epithelial ovarian cancer diagnosis resulted in survival bias, we performed additional analyses limited to BRCA1/2-associated epithelial ovarian cancer patients with a DNA test result before cancer diagnosis (n = 73 BRCA1; n = 9 BRCA2) and their matched sporadic controls. Results The median follow-up was 4.4 years (range 0.1–30.1). During the first three years after epithelial ovarian cancer diagnosis, progression-free survival was better for BRCA1 (HR 0.88, 95% CI 0.74–1.04) and BRCA2 (HR 0.58, 95% CI 0.41–0.81) patients than for sporadic patients. Overall survival was better during the first six years after epithelial ovarian cancer for BRCA1 (HR 0.7, 95% CI 0.58–0.84) and BRCA2 (HR 0.41, 95% CI 0.29–0.59) patients. After surviving these years, survival benefits disappeared or were in favor of the sporadic patients. Conclusion For epithelial ovarian cancer patients who received chemotherapy, we confirmed survival benefit for BRCA1 and BRCA2 germline pathogenic variant carriers. This may indicate higher sensitivity to chemotherapy, both in first line treatment and in the recurrent setting. The observed benefit appears to be limited to a relatively short period after epithelial ovarian cancer diagnosis.

Introduction and overall survival (OS) between either germline BRCA1-associated EOC patients or germline BRCA2-associated EOC patients and matched sporadic EOC patients treated with first-line chemotherapy.

Study population
For this retrospective matched cohort study, we selected BRCA1 and BRCA2 gPV carriers with a history of EOC from the national Hereditary Breast and Ovarian Cancer Netherlands (HEBON) database. In the context of the HEBON study, members of breast and/or ovarian cancer families are being identified through the departments of Clinical Genetics/Family Cancer Clinics at eight Dutch academic centers and the Netherlands Cancer Institute [35]. The study was approved by the Medical Ethical Committees of all participating centers. Written informed consent was obtained from each participant or from a close relative in case of deceased individuals. Relevant data on participants including data on preventive strategies, the occurrence of cancer, and vital status were retrieved and updated through medical files and questionnaires, and through linkages to the Netherlands Cancer Registry, the Dutch Pathology Database, and the municipal registry database. The latest follow-up date was December 31, 2017.
From this national cohort, we selected 389 BRCA1 gPV carriers and 123 BRCA2 gPV carriers with EOC. Patients were eligible for the study if they were diagnosed with EOC after 1988, had a proven BRCA gPV (with DNA test result either before or after EOC diagnosis), and received chemotherapy after diagnosis of primary EOC (in case of surgery, either before or after).
The selected BRCA gPV carriers were matched 1:1 to sporadic EOC patients from the National Cancer Registry on year of birth (+/-5 years), year of EOC diagnosis (+/-5 years), and FIGO stage (�IIA/�IIB/unknown). Sporadic patients were defined as patients who were either not DNA tested due to a negative family history of breast cancer or ovarian cancer or because DNA testing was not available yet, or DNA tested and without a proven BRCA gPV. Notably, about 5% of EOCs have a somatic BRCA pathogenic variant, but somatic testing has only been widely implied since 2020 in the Netherlands and data hereon is therefore not available for the current cohort.

Data collection
We retrieved data on the associated gene (i.e. BRCA1 or BRCA2) and date of DNA test result, dates of birth and death, and dates of diagnosis of EOC, first recurrent disease, and other cancers. We also collected data on tumor characteristics (FIGO stage, histology, and differentiation grade), CA125 at EOC diagnosis, and treatment details after primary EOC diagnosis and in the recurrent setting (surgery, type of chemotherapy, and maintenance treatment with poly (ADP-ribose) polymerase inhibitors (PARPi)).

Statistical analyses
We evaluated clinical characteristics by comparing EOC patients with (BRCA1 and BRCA2 groups) and without a proven BRCA gPV (sporadic groups). We used Pearson's chi-squared test for differences between the BRCA groups and the sporadic groups for categorical variables, and Wilcoxon rank-sum to test the equality of the medians for continuous variables.
The outcomes PFS and OS were measured in person-years of observation. The observation period started at the date of EOC diagnosis, and ended at the date of a censoring event or the date of first recurrence for the PFS analyses or death for the OS analyses. Censoring events included diagnosis of a new primary malignant tumor, date of last follow-up, and date of death (for PFS only).
To estimate the associations between gPV status (BRCA1 or BRCA2 versus sporadic) and survival endpoints, we used Cox proportional hazards models with the sporadic groups as the references to obtain hazard ratios (HR) with corresponding 95% confidence intervals (CI). We considered age at EOC diagnosis, grade, CA125 at diagnosis, type of chemotherapy, debulking surgery (yes/no), and complete debulking surgery (yes/no, i.e. the absence/presence of any residual disease) as potential confounders. The matching variables year of birth, year of EOC diagnosis, and FIGO stage were by definition not confounding factors. We generated Kaplan-Meier survival curves, and used the log-rank test for equality of survivor functions to test whether the curves were significantly different from each other. We performed all analyses separately for BRCA1 and BRCA2 gPV carriers.
Further, BRCA gPV carriers who underwent DNA testing after EOC diagnosis survived at least until this DNA test, which was in some cases many years after EOC diagnosis. To investigate whether this resulted in survival bias in favor of the BRCA gPV carriers, we also performed prospective analyses limited to BRCA-associated EOC patients with a DNA test result before EOC diagnosis and their matched sporadic controls.
Furthermore, as previous studies reported different short-term and long-term survival rates for gPV carriers [31][32][33], the proportional hazards assumption may be violated. Therefore we used Schoenfeld residuals to test whether the proportional hazards assumption is violated. If that was the case, we stratified the Cox models by a specified time-of-observation, i.e. the moment t where the HR switched from under 1 to above 1 (or vice versa). We calculated this exact moment using the formula where x is the variable of interest (i.e. BRCA1/BRCA2 or sporadic), β is the β coefficient, and δ is the time-varying coefficient. When the proportional hazards assumption is valid, δ equals zero. Otherwise, we can calculate t using where β and δ are derived from the Cox model including both the variable for gPV status and the interaction term of this variable with time. All p-values were two-sided, and a significance level α = 0.05 was used. Analyses were performed using Stata/SE (version 16.0, StataCorp, Collegestation, TX).

Study population
As shown in Table 1, the 389 BRCA1 gPV carriers and the 123 BRCA2 gPV carriers had longer follow-up than their matched sporadic EOC patients (median years 4.8 versus 3.5 for the BRCA1 comparison, p<0.001; 5.7 versus 3.5 for the BRCA2 comparison, p<0.001). The vast majority of the patients received platinum-based chemotherapy. After recurrence of disease, BRCA1-associated EOC patients were treated more often with chemotherapy than the sporadic patients (90% versus 79%, p<0.001), which did not apply for the BRCA2 comparison ( Table 1). The characteristics for the dataset used for the prospective analyses (in total n = 82 matched pairs) are shown in S1 Table.

Potential confounders
No differences between the groups were observed for the matching variables year of birth, year of EOC diagnosis, and FIGO stage, nor for age at EOC diagnosis and type of chemotherapy (Table 1). Due to the large proportion of missing data for CA125 at diagnosis, EOC grade, and

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Progression-free survival and overall survival after BRCA1/2-associated epithelial ovarian cancer completeness of debulking surgery, no adjustment was possible for these variables. We performed Cox models adjusted for debulking surgery (yes/no), with the sporadic groups as the references.

Survival analyses
We observed no differences between the BRCA-associated groups and their matched sporadic patients in the percentage of patients with recurrent disease. The time between diagnoses of EOC and first recurrence, though, was longer for BRCA-associated patients than for the sporadic patients ( Table 2). Accompanying survival curves are depicted in   (Table 2). Overall, as shown in Table 3, BRCA2 gPV carriers showed better PFS (HR 0.67, 95% CI 0.5-0.91) and OS (HR 0.61, 95% CI 0.44-0.83) than their matched sporadic patients, which can also be seen in Fig 2. The stratified analyses revealed a significant risk-reduction in favor of BRCA2 gPV carriers for PFS (HR 0.58, 95% CI 0.41-0.81; Table 3) and OS (HR 0.41, 95% CI 0.29-0.59) for the observation period under t (being 3 and 6 years, respectively), but a higher risk for death after t (HR 3.14, 95% CI 1.34-7.34). The numbers of patients in the prospective analyses were too small to draw meaningful conclusions (Table 3 and Fig 2).

Discussion
In this retrospective matched cohort study, we confirmed better PFS during the first three years after EOC diagnosis and OS benefit during the first six years for patients with a BRCA1 or BRCA2 germline PV. After surviving this period, the benefit disappears, and might even turn into a higher risk of dying for gPV carriers. The observed survival benefit was slightly stronger for BRCA2 than for BRCA1.
Our results are in line with a number of previous studies. Studies with limited follow-up periods showed improved PFS and OS-with comparable periods without progression and time till death as seen in our study-for BRCA1-associated EOC patients [23], BRCA2-associated patients [23,30], or combined BRCA1/2 cohorts [10,22,[24][25][26]. Studies with long-term periods of follow-up showed that improved overall survival seems to be mainly driven by the   first five years after diagnosis, with no benefit for those surviving that first period [31,33,36], or even worse OS afterwards [32], as observed in the current study. Previously observed survival benefit could have been an age-effect. Recently, Mallen et al. observed worse survival for older patients, although the authors noted this may merely be the result of tumor biology rather than comorbidities [37]. As we currently matched-indirectly by matching on year of birth and year of diagnosis-on age at diagnosis, in contrast to most of the previous studies, our results support the suggestion that the observed difference is not related to age.
Our results support the hypothesis regarding BRCA1/2-associated EOC patients being more sensitive to platinum-based chemotherapy, especially since none of the patients in the current cohort received first-line maintenance treatment with a PARPi. Primary systemic treatment was not different for EOC patients with and without a BRCA1/2 germline gPV. Therefore, differences in PFS cannot be attributed to differences in received chemotherapy treatment, leaving gPV status as the most likely explanation. Although debulking surgery was performed in the vast majority of the patients (~95%), sporadic patients underwent less often debulking surgery, possibly due to a very poor prognosis of disease at diagnosis, or due to the presence of comorbidities. As this may indicate a higher baseline risk for death in the sporadic EOC groups, we adjusted the analyses for this variable.
In the recurrent setting BRCA-associated EOC patients were more often treated with chemotherapy, which may have influenced survival. The rationale for omitting systemic treatment may have been a worse clinical situation at presentation of recurrent disease, potentially resulting in a higher baseline risk of dying after recurrent EOC in the sporadic group. Further, in the BRCA groups more patients received PARPi after recurrent disease as a maintenance therapy. However, since PARPi has only been administered since 2015, the majority of the patients in the current cohort (approximately 95%) did not receive PARPi. For the sake of completeness, we performed subgroup analyses for OS excluding patients who were treated with PARPi in the recurrent setting and their matched counterparts. As none of the patients were treated with PARPi in the first-line, such subgroup analyses were not necessary for PFS. The subgroup analyses for OS revealed similar results as the original analyses (HR 0.81, 95% CI 0.68-0.97 for BRCA1 and HR 0.65, 95% CI 0.47-0.9 for BRCA2). Thus, the influence of PARPi in the recurrent setting is very limited in this study. This will increase, though, in future studies due to current clinical practice [38].
As previously described, including prevalent cases in studies involving BRCA gPV carriers can introduce seriously biased results [39,40]. The majority of the gPV carriers in our cohort had their DNA tested after EOC diagnosis, with survival times up to 20 years until DNA test. Reassuringly, for the BRCA1 comparison, the additional prospective analyses showed comparable overall survival as for the complete analyses, suggesting minimal bias as a result from delayed DNA testing. Unfortunately, due to the small number of BRCA2 gPV carriers with a DNA test before cancer diagnosis, we were unable to draw meaningful conclusions from the prospective analyses among BRCA2 gPV carriers and matched sporadic patients. Alternatively, we performed left-truncated analyses with the observation for the BRCA groups starting at the date of either DNA test result or EOC diagnosis, whichever came last, thus excluding patients with recurrent disease, LFU or death before DNA test result. As shown in S2 Table, the results were comparable to those for the complete and prospective analyses, confirming minimal bias due to delayed DNA testing.
Other strengths of the current design include the separate BRCA1 and BRCA2 analyses, and the fact that we matched-indirectly by matching on year of birth and year of diagnosis-on age at diagnosis. The advantage of the latter is that also the sporadic EOC patients were relatively young at diagnosis. Therefore, we can reasonably assume that the leading cause of death is ovarian cancer in both groups, and that the mortality in the sporadic group is not distorted by competing causes of death due to older age.
One of the limitations of the study may be that not all sporadic patients were tested for a BRCA germline gPV. As mentioned before, the majority of the BRCA-associated EOC patients were tested after cancer diagnosis. Theoretically, the sporadic group may contain patients who were actually gPV carriers but never had the opportunity to get tested because they had died before DNA testing was performed or was even implemented in clinical practice. Unintentional misclassification of deceased patients in the sporadic groups may simultaneously overestimate the risk of dying in the sporadic groups and underestimate that risk in the BRCA groups, thus potentially overestimating the benefit for BRCA gPV carriers. Oppositely, the sporadic group may also include untested gPV carriers without recurrent disease or death, oppositely leading to an underestimation of the benefit. With regard to potential misclassification, we would like to emphasize that with the introduction of PARPi in 2015, more and more EOC patients undergo DNA testing sooner after diagnosis in order to receive the optimal treatment, at first only in the recurrent setting but nowadays also at primary disease.
In addition, data on somatic testing is not available for the current cohort. As a result, the sporadic group may contain a number of BRCA positive specimens, which may have influenced the results. However, as about only 5% of EOCs have a somatic BRCA pathogenic variant, we think this influence will be limited. Moreover, under the assumption that survival benefit will also apply to EOCs with a somatic BRCA pathogenic variant, potential misclassification of these EOCs in the sporadic group would led to an underestimation of the observed survival benefit on the short-term rather than an overestimation. Therefore, although the lack of data on somatic testing may be a deficiency in the study, in our opinion this may play a minor role.
Another limitation is the limited availability of data regarding complete debulking or residual disease after primary surgery in our study. In a previous study the only independent prognostic factor for survival in BRCA1/2 gPV carriers was the extent of debulking at primary surgery, with better survival for patients without macroscopic disease [10]. Recently, Ataseven et al. confirmed that complete macroscopic tumor resection is a strong prognostic factor in patients with EOC, regardless of BRCA status [26]. In the current study we did adjust for debulking surgery (yes/no), but the amount of residual disease may be more important in this respect. However, we found no differences between the comparison groups in the percentages of patients with residual disease for those patients with available data. Therefore, we expect no influence on the estimated HRs by adjusting for the amount of residual disease.
In conclusion, in this large case-matched cohort study we confirmed survival benefit for BRCA1/2-associated EOC patients treated with mainly platinum-based chemotherapy. This may indicate higher sensitivity to chemotherapy, both in the first-line and in the recurrent setting. The observed benefit appears to be limited to a relatively short period after EOC diagnosis. Future research is warranted to assess in more detail the added value of PARPi on both PFS and OS, especially on the long-term, where the benefit of classic systemic treatment seems to diminish and even disappear.
Supporting information S1